Research Structure Background

"Prevention and Control of Avian Influenza in the U.S." Research Structure

ACCOMPLISHMENTS OF THE PREVIOUS 3 YEARS

The biological significance of influenza viruses and its major economical importance for the poultry and swine industry as well as its implications for public health were discussed in the submission of AICAP 1. In order to elaborate more on the preliminary data and the future of the AICAP for the next three years, we have kept the background and significance section as brief as possible.

BACKGROUND AND SIGNIFICANCE

B1. Influenza viruses. Influenza A viruses are divided into subtypes based on the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins(39). To date, 16 HA and 9 NA subtypes have been identified(103). The segmented nature of the virus genome, which consists of eight different molecules of single stranded RNA, allows reassortment of genes when a susceptible host is co-infected with two different influenza virus subtypes. Influenza viruses with many combinations of HA and NA subtypes are routinely isolated from birds(71).

B2. Interspecies transmission and virulence determinants in influenza virus. Wild aquatic birds (Orders Anseriformes and Charadriiformes) are considered the primary hosts of influenza A, in which the virus is enzootic(103). In these birds, influenza viruses usually replicate in the intestinal tract, cause no disease, and spread by fecal contamination of the water habitat(77). These viruses occasionally infect land-based birds (Order Galliformes) causing epizootics. In land-based poultry, influenza A infections range from asymptomatic to lethal. Virulence for chickens and other poultry can only result from infections with influenza A viruses that replicate efficiently in these species; i.e. that have crossed the species barrier. Viral determinants of virulence consist of individual viral components interacting with one another and with elements of the host cell, the immune system of the host, and the environment(1, 65, 109). The importance of efficient replication and spread of influenza A virus in a new host, such as land-based poultry, cannot be overemphasized; it is a pre-requisite for the expression of virulence. Multiple genes that determine replication efficiency are critical determinants of host range.

The cleavability of the HA protein plays a critical role in the pathogenicity of avian influenza viruses because it restricts tissue tropism(29, 30, 35, 36, 68, 82, 95). All highly pathogenic avian influenza viruses (HPAI) identified to date differ from their low pathogenic (LPAI) counterparts in the susceptibility of the HA to host proteases. HPAI are characterized by HAs that are highly susceptible to cleavage by numerous host proteases. In contrast, LPAI HA requires specific active proteases, such as trypsin, for cleavage and activation of infectivity.

Mortality rates with extremely virulent HPAI viruses may be as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all viruses of these subtypes are necessarily HPAI(89). Viruses with any other subtype cause a mild, primarily respiratory disease in poultry, which may be exacerbated by other infections or environmental conditions. Unlike other HA subtypes, the highly virulent H5 and H7 viruses possess multiple basic amino acids at the cleavage site of the HA. Since 1959, 21 primary outbreaks of HPAI in poultry have been reported, with 12 primary outbreaks since 1990. Since 2003, the continued circulation in Asia of HPAI of the H5N1 subtype, concomitantly with the circulation of other LPAIs of different subtypes represents a major challenge in terms of understanding the factors that contribute to their perpetuation as well as to devise strategies aimed at eradicating these viruses from domestic poultry. There is certainly a sense of urgency in achieving these goals since many of these Asian viruses have shown an unprecedented expansion of their host range with yet to be determined major ecological consequences. Most notably the ability of these viruses to infect humans accompanied by a high mortality rate suggests that a pandemic is looming over the global community with potentially devastating consequences.

B3. Agricultural practices contributing to infection with influenza. 'Biosecurity is the first line of defense against all avian influenza viruses. Preventing the introduction of avian influenza by eliminating all contact between commercial poultry and wild birds, swine farms, and live bird markets (LBMs) is a common, routine and successful practice' (USAHA 2002 Resolution N 28). Unfortunately, implementation of strict biosecurity measures in the LBM systems around the world is difficult because of environmental risks factors and cultural practices that contribute to the perpetuation of viruses in these marketing systems(16, 75). Intensive education programs are necessary to make owners, suppliers, wholesalers, dealers of commercial poultry and the general public aware of the threat of avian influenza not only for poultry but also potentially to humans. Most efforts at controlling avian influenza in poultry have been focused on the H5 and H7 subtypes; however, control measures should arguably be in place to diagnose and control influenza viruses regardless of the HA subtype. In this regard the AICAP 1 made a major contribution and developed important education programs for this segment of the poultry production chain as described in more detail in the Preliminary Data section. In addition, many of the biosecurity and control measures for avian influenza can help to reduce the spread of other poultry diseases.

B4. Diagnostic tools and vaccines against avian influenza.

The diagnosis of avian influenza is straightforward: virus isolation in eggs and agar gel immunodiffusion assay, although lengthy, are considered the gold standards(17, 19, 73, 79, 98). Real time reverse transcription PCR (RRT-PCR) has been used for the detection of influenza and it was adopted by NVSL as a key diagnostic test(79). A great advantage of this system is that it can be modified to detect influenza viruses of all known subtypes and/or specific subtypes depending on the need. Taking advantage of the integrated nature of this proposal, we intend to develop, validate, and implement, in diagnostic laboratories across the country, more accurate, sensitive, reproducible and simpler tests specifically designed for early detection of avian influenza. We will concentrate on techniques to identify the HA subtypes that have been associated with major economic losses in poultry in the US and abroad.

Vaccination is the second line of defense against avian influenza. The USDA has licensed few inactivated virion avian influenza vaccines and a recombinant fowlpox-avian influenza H5 HA vaccine(27, 86-88, 106). Much needs to be done with respect to effective influenza vaccines since the currently approved methods are limited in terms of their protective efficacy in birds other than chickens. For example, little is known about the protective efficacy of current vaccines against AI in ducks; although initial tests have been rather disappointing and underscore the need to develop more effective vaccines. Further studies are needed to evaluate vaccine effectiveness in viral shedding and transmission and, more importantly, the generation of mucosal immunity(59, 84, 85, 90, 100). Taking advantage of the collaborative nature of this project, we propose to develop and test different vaccination strategies and run pilot tests to assess their efficacy, potency and evaluate their performance to decrease or eliminate viral load.

PRELIMINARY RESULTS TOP

During the past 2 1/2 years AICAP 1 made significant impacts in all areas of the original proposal. A bullet list of accomplishments that stand out for each specific area is highlighted below. Further information is found in the appendix.

Molecular basis for adaptation of influenza A viruses from wild aquatic birds and/or intermediate hosts to land-based poultry. AICAP 1 investigators demonstrated the role of quail (Coturnix coturnix) as an intermediary host that can change and expand the host range of avian influenza viruses. In addition, AICAP investigators showed that the respiratory and intestinal tract of quail presents abundant human-like sialic acid receptors, which could partically explain the emergence of influenza strains with the capacity to infect humans. AICAP 1 investigators performed also an initial characterization of swine influenza viruses of the H3N2 subtype that caused outbreaks in turkeys in the US.

Dynamics and evolution of influenza A viruses in waterfowl of the four major flyways of the US: Contribution to the emergence and perpetuation of avian influenza in land-based poultry. Led by Dr. Richard Slemons at Ohio State University, the AICAP 1 assembled the first continent wide network to study the ecological and biological characteristics of AI viruses isolated from wild-birds. The work has involved also the research arm of USDA (ARS) as well as cost-sharing institutions across the country. The participation of AICAP investigators in several of the newly formed NIAID-NIH centers of excellence in influenza research and surveillance emphasizes the important role that AICAP played in situating key AICAP members in other research network structures. A summary of research results is presented in the appendix.

Effective education and biosecurity programs. A hallmark of AICAP 1 was the integration of research and education programs. The AICAP adopted a strategy to channel research findings into an audience that would directly benefit from our efforts. In this regard the AICAP is unique and we must emphasize that neither the recently established NIAID-NIH centers of excellence in influenza research or other federal programs have integrated research and education programs as we do. While many education and training opportunities exist for poultry producers to learn about AI and preventative steps to keep flocks from infection, no such opportunities were available for game bird producers and hunting and shooting preserve owners. Yet game birds may be an important link between AI viruses that infect primarily waterfowl and those that become adapted to poultry. The North American Gamebird Association (NAGA) has over 3,000 members representing every state and seven countries. Farms with over 100,000 animals and many with more than 200,000 are common, representing a $4 billion industry. The AICAP developed education programs to provide game bird breeders information on AI, biosecurity steps to prevent infection and training in necropsy, serological sample taking and steps to becoming NPIP AI Clean according to subpart E. In addition to the producers, extension veterinarians, practicing veterinarians and extension educators also receive training to continue working with their game bird clientele. Organized by Dr. Eva Wallner-Pendleton at Penn State University, the AICAP program conducted game bird health workshops/AI and biosecurity presentations in 12 states across the US with the participation of additional collaborators as shown below: California, Drs. Cardona, Smith, Bland; South Carolina, Drs. Helm, Kelly, Hall; Tennessee, Dr. Cartwright; Alabama, Drs. Giambrone, Blake, Hoerr; Connecticut, Drs. Khan, Dunn; Florida, Dr. Cartwright; Pennsylvania, Drs. Hulet, Patterson; Washington State, Drs. Dhillon, Cardona, Reimers; Kansas, Dr. Pendleton; South Dakota, Dr. Pendleton; Texas, Drs. El-Atrache, Cartwright; Minnesota, Drs. Halvorson, Shaw, Zeigler. Surveys were performed at the end of the training sessions. A Powerpoint presentation with a summary of the deliverables and survey results is included in the appendix.

Educational program on in-house depopulation and composting methods for use during catastrophic mortality or depopulation. A comprehensive training program was developed by George Malone at the University of Delaware and Dr. Nathaniel Tablante at the University of Maryland, College Park to prepare responders on the options for depopulation and disposal of AI-infected meat-type poultry flocks. The half-day comprehensive training program covers human health considerations when responding to an AI outbreak and the options for depopulation and carcass disposal of infected breeder, broiler and turkey flocks. Response to this training has been highly favorable and very timely. Based on pre- and post-training questionnaires, participants identified depopulation procedures and the in-depth instructions on in-house composting for carcass disposal and virus containment as being the most helpful. Since its inception the program has conducted 33 training sessions across the country in the following locations: Millsboro, DE; Moorefield, WV; Harrisonburg, VA; Statesville, NC; Lumberton, NC; Mt. Olive, NC; Lancaster, PA; Columbia, SC; APHIS/Riverdale, MD; Athens, GA; Montgomery, AL; Nashville, TN; Princeton, KY; Fayetteville, AR; Hope, AR; Ruston, LA; Kelso, WA (for WA and OR); Minneapolis, MN; Magee, MS; Bryan, TX; Tulsa, OK; Columbus, OH; Celina, OH; Raleigh, NC; Storrs, CT; Tallahassee, FL; Modesto, CA; Seattle, WA; Washington DC; Willmar, MN; Newark, NJ; Sedalia, MO; West Lafayette, IN. The training program has been very successful and requests were made to conduct similar training sessions abroad, which were carried out with no cost to the AICAP, in Canada (5 sessions covering 4 provinces, Quebec, Ontario, Alberta and British Columbia) and Brazil (2 sessions). A summary of the training program and questionnaire results is included in the appendix.

Development of critical diagnostic tests: A crucial element in the control of AI outbreaks is the early identification of flocks infected with avian influenza. The earlier an infected flock is identified and quarantined, the less chance of the virus spreading from that flock will occur and the outbreak can be contained faster and at lower cost. Although we have tests for the direct detection of AI as well as serologic tests, for the eradication phase of a control effort, the direct detection methods are the most important for controlling the disease. To complement the diagnostic toolkit available to the poultry health professional, AICAP investigators Drs. Vikram Vakharia at the University of Maryland Biotechnology Institute and Daniel R. Perez at the University of Maryland, College Park helped develop in cooperation with Dr. Chinta Laminchhane at Synbiotics Co. a rapid antigen capture test, Flu DetectTM, which is rapid, sensitive, inexpensive, and can be performed penside. Similar to a simple pregnancy test, the easy-to-use kit has been selected by the FAO (Food and Agriculture Organization, United Nations) for use in their multinational initiative to monitor and control the spread of infectious avian influenza (http://www.worldpoultry.net/news/id2205-11642/flu_detect_selected_by_fao.html). The antigen-capture test and a complementary antibody ELISA detection kit (ProFlok AIV ELISA kit) have received USDA approval and have attracted an important portion of the international market.

Development of novel vaccine strategies. The goal of AICAP investigators was to develop alternative vaccine strategies that would be more effective than current approaches and amenable for mass vaccination. In this regard two groups within the AICAP were successful in developing live recombinant vaccines that show great promise as vaccines for mass immunization. Further alternative approaches will be discussed in the research design and methods section.

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RESEARCH DESIGN AND METHODS